Transcription profiling by array of ESC derived hepatocytes treated with either vehicle or high energy substrates to model non alcoholic fatty liver disease
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ABSTRACT: Female H9 ESCs were maintained on matrigel and differentiated into hepatocyte like cells as previously described (Szkolnicka D, Farnworth SL, Lucendo-Villarin B, Hay DC. Curr Protoc Stem Cell Biol. 2014 Aug 1;30:1G.5.1-12. doi: 10.1002/9780470151808.sc01g05s30) . Sodium L-Lactate (L7022, Sigma, Gillingham, UK) 10mM, Sodium Pyruvate 1mM (Sigma, Dorset, UK) and Octanoic Acid (C2875, Sigma, Gillingham, UK) 2mM for 48 hours before harvesting.
Project description:This experiment is designed to compare the effects of Vitamin D3 treatment of mouse bone marrow derived dendritic cells with control, untreated cells, and additional effects of maturation of either treated or control cells with LPS.
Project description:Stable clones of RPE-1 cells expressing tetracycline-inducible wild type SAS-6 or SAS-6ND were obtained via lentiviral gene transduction with the pLVX tet-on Advanced inducible gene expression system (Clontech). Stable expressors were derived by selection with 5 μg/mL puromycin (Sigma-Aldrich, UK). Doxycycline 1 microgram/mL was added to growth media for 6 days to induce SAS-6 expression. RNA was isolated from RPE-1 cells using the RNeasy mini kit (Qiagen, CA) according to the manufacturer’s protocol. Gene expression was profiled using GeneChip™ Human Transcriptome Array 2.0.
Project description:Chronic metabolic acidosis occurs commonly in patients with diseased kidneys and is linked with progression to renal failure. As renal function declines there is loss of nephron mass and adaptive proximal tubular hypertrophy and hypermetabolism. We have previously demonstrated in an in vitro model of chronic acidosis in porcine LLC-PK1 cells increased ammonia generation (oxidative hypermetabolism) linked with tubular cellular dysfunction and increased markers of oxidative stress. The aim of this study was to determine the effect of chronic acidosis in HK-2 cells on changes in gene and protein expression stimulated by oxidative stress. HK-2 cells were were seeded onto 6 well plates at a density of 4x104 cells/well and cultured for 72 h in growth medium (DMEM/Ham s F12 medium supplemented with 5.5 mM glucose, 2 mM L-glutamine, 5 ug/ml insulin, 5 ug/ml transferrin, 5 ng/ml sodium selenite, 0.4 ug/ml hydrocortisone, 5 ng/ml epidermal growth factor, 100 U/ml penicillin, 100 ug/ml streptomycin, and 10% FCS). The medium was then replaced by growth media buffered to pH 7.4, 7.0 or 6.7 with 12.5 mM bis-Tris and cells were cultured for further 48 h. Media were changed to serum free media (SFM) buffered by 12.5 mM bis-Tris to pH 7.4, 7.0 or 6.7 and cells were harvested and RNA isolated after 24 h. Total RNA was isolated from HK-2 cells on 3 different occasions using Tri-reagent (Sigma, UK) as recommended by the manufacturer's protocol. The RNA from the respective replicate samples were pooled together and the RNA content was measured using RiboGreen RNA Quantitation Reagent (Molecular Probes, Invitrogen, UK). The integrity of the total RNA was verified on a denaturing agarose-formaldehyde gel.
Project description:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ which can be modelled using primary cells in vitro provides the opportunity for selected genetic effects to be investigated in detail.
Project description:TNFalpha and IL1beta play a pathogenic role in rheumatoid arthritis. Both cytokines are known to activate cytokine and metalloproteinase secretion by synovial fibroblasts. In the present study, we wanted to investigate whether TNFalpha and IL1beta displayed differential effects on cultured Fibroblast-like Synovial Cells derived from RA patients. Global gene expression analyses indicated that both cytokines induced similar genes in these cells. Fibroblast-like cells (FLS) were purified from synovial biopsies from RA patients. Briefly, minced synovial fragments were digested in 1 mg/ml hyaluronidase solution (Sigma Aldrich, St Louis, MO) for 15 minutes at 37M-BM-0C and 6 mg/ml collagenase type IV (Invitrogen, Paisley, UK) for 2 hours at 37M-BM-0C. Next, cells were washed, resuspended in high-glucose DulbeccoM-bM-^@M-^Ys Modified Eagle Medium (Invitrogen) supplemented with 1% antibiotics-antimycotics (Invitrogen) and 1% MEM sodium pyruvate (Invitrogen), and seeded at 10,000 cells/cm2 in 6-well plates. When the cells reached confluence, adherent cells were detached using sterile 0.5% trypsin-EDTA (Invitrogen) and used as FLS between passages 3 and 9. Cells were seeded in 24-well plated at 25.000/well and incubated overnight with or without the following cytokines : TNF-M-NM-1 (R&D Systems, Minneapolis, MN) 10 ng/ml, IL-1M-NM-2 (R&D Systems) 10 ng/ml. After overnight incubation with the indicated cytokines, cells were harvested and total RNA was extracted using the NucleospinM-BM-. RNA II extraction kit (Macherey-Nagel, DM-CM-<ren, Germany), in order to be labeled according to a standard Affymetrix procedure and hybridized on HGU133 Plus 2.0 Human Genome slides.
Project description:Monobromobimane (mBBr) labelling: mBBr labelling was carried out either immediately prior to 42°C, two hour heat shock, or immediately following heat shock. Medium was removed from culture flasks and cells were washed using PBS. Cells were then labelled by incubation at 37°C with 6 mL of 400 µM mBBr (Sigma-Aldrich, #B4380) for 10 minutes. Following labeling, 6 ml of 2 mM L-glutathione reduced (GSH, SigmaAldrich, #G4251) in PBS was added to quench the mBBr reaction. The quenched mBBr solution was removed and cells were washed with PBS. Mass spectrometry (MS) sample preparation, analysis and data processing: Six 1.6 mm steel beads (Next Advance Inc.) were added to the cell pellet tube with 30 µL SL-DOC (1.1% (w/w) sodium dodecyl sulfate (Sigma), 0.3% (w/w) sodium deoxycholate (Sigma), 25 mM ammonium bicarbonate (AB, Fluka), in de-ionised (DI) water containing 0.1% (v/v) protease inhibitor cocktail (Sigma), and 0.1% (v/v) phosphotase inhibitor cocktail (Sigma). Cells were homogen
Project description:Differentially expressed genes in the stomachs of type-1 gastric neuroendocrine tumour (NET) patients before, during and after treatment with netazepide (YF476)
Project description:We collected up to 6 separate analytes per patient from 14 HNSCC individuals and evaluated spatial (9 cases) and/or temporal (8 cases) variability using RNAseq. Sections taken from a frozen section using a crytostat were analysed for RNA quality was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies UK Ltd., Stockport, UK). Total RNA was converted into a library for sequencing on the HiSeq 2000 (Illumina Inc., San Diego, USA) using the TruSeq⢠stranded mRNA Sample Preparation Kit (Illumina Inc.). Briefly, poly-A mRNA was purified from total RNA (100ng) using the Poly(A) Purist Mag Kit (Life Technologies Ltd., Paisley, UK), according to the manufacturerâs instructions. The mRNA was then amplified and converted into cDNA, which was purified and used to construct libraries that were hybridized to the flow cell for single end (SE 35bp) sequencing. The protocol employed yielded 35-bp long reads. The quality of raw SE read data in FASTQ files were assessed and reads of low quality were trimmed or removed. SE reads were then mapped to the human genome (hg19) using TopHat (version 2.0.9) and, following the removal of multi-mapping reads, converted to gene specific read counts for annotated genes using HTSeq-count (version 0.5.4).
Project description:Human PdLFs (iCell Bioscience, China) were treated with 1µM, 10µM, and 100µM L-lactic acid (Sigma-Aldrich, Germany). Phorbol 12-myristate 13-acetate (PMA) (ab120297, Abcam, UK) at 1µM was used as an inflammatory inducer, a positive control for the expression and synthesis of collagen and MMP-1. Untreated PdLFs were regarded as control. PdLFs were incubated at 37°C and 5% CO2 for 2 days and 6 days.